Figure 1.
Stimulation of NO synthesis by glucose in mice liver membrane and its inhibition by NAME.
The liver membrane in HEPES buffer, pH 7.4, was prepared from the liver of adult mice as described in the Materials and Methods and incubated with different amounts of glucose in the presence of 2.0mM CaCl2 in a total volume of 1.0ml for 30 min at 37°C. The synthesis of NO was determined by methemoglobin method as described in the Materials and Methods. Identical reaction mixtures contained 0.1mM NAME were incubated under identical conditions and the synthesis of NO was similarly determined.
Each point represents mean ± S.D. of 5 experiments by using 5 different animals each in triplicate.
The solid circles (●) represent the synthesis of NO in the liver membrane preparation in the presence of glucose alone, the solid squares (■) represent the synthesis of NO in the liver membrane preparation in the presence of both glucose and NAME.
Figure 2.
Line-Weaver Burk plot of the stimulation of glucose induced NO synthesis in mice liver preparation.
The mice liver membrane in HEPES buffer, pH 7.4, was incubated in the presence and absence of 0.02M glucose with different amounts of l- arginine and 2.0mM CaCl2 in a total volume of 1.0ml. After incubation for 30 min at 37°C the synthesis of NO was determined by methemoglobin method as described in the Materials and Methods.
Line A represents the synthesis of NO in the absence of added glucose to the reaction mixture with different concentrations of l-arginine. Line B represents the synthesis of NO in the reaction mixture in the presence of 0.02M glucose and with different amounts of l-arginine.
Figure 3.
Time course of glucose uptake by the GLS in the presence and absence of NAME.
The grated mice liver suspension (GLS) in HEPES buffer pH (7.4), was prepared as described in the Materials and Methods. Typically, 30-40 mg of GLS in the HEPES buffer, pH 7.4, was incubated in the presence of 0.02M nonmetabolizable 2-deoxy-D- glucose with 1µl of the 14C-labelled sugar as described in the Materials and Methods for different times as indicated. In parallel experiments, 0.1mM NAME was added to the identical reaction mixture and glucose uptake in both cases was determined.
Results are mean ± S.D. of 5 experiments each in triplicate using GLS preparation from 5 different animals.
Solid circles (●) represent the uptake of glucose by GLS in the presence of 0.02M glucose and solid squares (■) represent the uptake of glucose by GLS in the presence of both glucose and NAME in the reaction mixture respectively.
Figure 4.
The appearance of Glut-4 on membrane peripheries of hepatocytes incubated with glucose with and without NAME.
The grated liver suspension in HEPES buffer, pH 7.4, was incubated with 0.02M glucose in the presence and absence of 0.1mM NAME in the incubation mixture for 30 min at 37°C. After incubation GLS “chunks” were sliced into 6-8 nm sections by using a cryostat. The sliced sections were treated with Glut-4 antibody to demonstrate the presence of Glut-4 by immunohistochemistry as described in the Materials and Methods.
Panel-A: The immunohistochemistry of liver sections was incubated in the presence of 0.02M glucose. Glut-4 in the liver cells was determined by using fluorescent tagged Glut-4 antibody. White arrow indicates the translocation of Glut-4 transporter to the periphery of liver cell membrane.
Panel-B: The immunohistochemistry of liver sections was incubated in the presence of both 0.02M glucose and 0.1mM NAME by using fluorescent tagged Glut-4 antibody as described in the case of panel-A. White arrow indicates the absence of translocation of Glut-4 transporter to the membrane periphery.
The figures shown are typical representative of six different experiments using 6 different animals.
Figure 5.
Synthesis of Glut-4 in GLS incubated either with different amounts of glucose or with NO.
Typically 30 to 40 mg of GLS in HEPES buffer, pH 7.4, was incubated with different concentrations of glucose (0-0.05M) as shown or in the presence of 2.5nmol NO solution in 0.9% NaCl or in the presence of both 0.02M glucose and 2.5nmol NO solution for 30 min at 37°C. In parallel experiments, 0.1mM NAME was added to the reaction mixture containing 0.02M glucose. After incubation for 30 min at 37°C, the amounts of Glut-4 produced in the reaction mixture were determined by ELISA using Glut-4 antibody. The amount of Glut-4 produced in the reaction mixture was determined by expressing the increase of the arbitrary optical densities at 405 nm.
▓ - Glut-4 synthesis in the presence of varying amounts of glucose; ≡ - Glut-4 synthesis in the presence of both glucose and NO; □- synthesis of Glut-4 in the presence of only NO; ■- Glut-4 synthesis in the presence of glucose and NAME.
Results shown are mean ± S.D. of the optical densities obtained from 6 experiments by using 6 different animals.
Figure 6.
Immunoblot analysis of Glut-4 synthesis by in vitro translation of Glut-4 mRNA in the GLS.
The grated liver suspension in HEPES buffer, pH 7.4, was incubated with different concentrations of glucose for 30 min at 37°C. After incubation, nucleic acids which contained mRNA of Glut-4 were extracted and translated in vitro as described in the Materials and Methods. The reaction supernatants were subjected to immunoblot analysis using Glut-4 antibody (Panel-A), the immunopositive bands were quantitated by using Image-J program by computer analysis. The integrated area of each band was also calculated (Panel-B).
Panel-A: Immunopositive bands of Glut-4 synthesis in GLS incubated in the presence of different concentrations of glucose in the incubation mixture as indicated.
Panel-B: Integrated area of each of the immunopositive band as shown in the panel-A.
Both Panel-A and Panel-B are the representative of experiments by using 6 different animals.
Figure 7.
Role of NO in insulin synthesis in glucose treated GLS via the expression of proinsulin genes.
The grated liver suspension in HEPES buffer, pH 7.4, was incubated with different amounts of glucose as indicated for 30 min at 37°C. After incubation, nucleic acids were extracted and insulin mRNA was translated in vitro as described in the Materials and Methods. The amount of insulin synthesized, was quantitated by ELISA using anti-insulin antibody. In parallel experiments, the incubation mixture was incubated with glucose and 0.1mM NAME and the synthesis of insulin was similarly determined.
Panel-A: Glucose induced synthesis of insulin in the presence and absence of NAME. The solid circles (●) represent insulin synthesis in the presence of different amounts of glucose and the solid squares (■) represent the synthesis of insulin in the presence of both glucose and 0.1mM NAME.
Panel-B: Agarose gel electrophoresis of cDNA prepared from the isolated insulin mRNA.
‘1’ and ‘2’ represents the expression of proinsulin genes I and II in the presence and absence of 0.02M glucose respectively.
Results shown are representatives of the optical densities obtained from 6 different experiments using 6 different animals.